molecular cloning methods Lecture Note.ppt
- 1. Molecular Biology Fourth Edition Chapter 4 Molecular Cloning Methods Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
- 2. 4-2 4.1 Gene Cloning • Gene cloning links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hosts • One can then produce large quantities of these genes in pure form
- 3. 4-3 The Role of Restriction Endonucleases • Restriction endonucleases, first discovered in the late 1960s, are named for preventing invasion by foreign DNA by cutting it into pieces • These enzymes cut at sites within the foreign DNA instead of chewing from the ends • By cutting DNA at specific sites they function as finely honed molecular knives
- 4. 4-4 Naming Restriction Endonucleases Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind III – First letter is from the genus H from Haemophilus – Next two letters are the 1st two letters of the species name in from influenzae – Sometimes the strain designation is included “d” from strain Rd – If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind I – If more than one restriction enzyme is produced, the others are numbered sequentially II, III, IV, etc.
- 5. 4-5 Restriction Endonuclease Specificity Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence – These enzymes can recognize 4-bp, 6-bp, 8-bp sequences – The frequency of cuts lessens when the recognition sequence is longer
- 6. 4-6 Restriction Enzyme Terminology • A 6-bp cutter will yield DNA fragments averaging 4000-bp or 4 kilobases (4kb) in length • Heteroschizomers recognize the same DNA sequence but use a different cutting site – they are also called isochizomers • These enzymes cut DNA strands reproducibly in the same place, which is extremely useful in gene analysis
- 7. 4-7 Use of Restriction Endonucleases • Many restriction endonucleases make staggered cuts in the 2 DNA strands – This leaves single-stranded overhangs, called sticky ends that can base-pair together briefly – This makes joining 2 different DNA molecules together much easier • Staggered cuts occur when the recognition sequence usually displays twofold symmetry, palindromes
- 8. 4-8 Restriction-Modification System • What prevents these enzymes from cutting up the host DNA? – They are paired with methylases – Theses enzymes recognize, methylate the same site • Together they are called a restriction-modification system, R-M system • Methylation protects DNA, after replication the parental strand is already methylated
- 9. 4-9 An Experiment Using Restriction Endonuclease • An early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosome • Each plasmid had 1 site for EcoRI – Cutting converted circular plasmids into linear DNA with the same sticky ends – The ends base pair • Some ends re-close • Others join the 2 pieces • DNA ligase joins 2 pieces with covalent bonds
- 10. 4-10 Summary • Restriction endonucleases recognize specific sequences in DNA molecules and make cuts in both strands • This allows very specific cutting of DNAs • The cuts in the two strands are frequently staggered, so restriction enzymes can create sticky ends that help to link together 2 DNAs to form a recombinant DNA in vitro
- 11. 4-11 Vectors • Vectors function as DNA carriers to allow replication of recombinant DNAs • Typical experiment uses 1 vector plus a piece of foreign DNA – Depends on the vector for its replication – Foreign DNA has no origin of replication, the site where DNA replication begins • There are 2 major classes of vectors: – Plasmids – Phages
- 12. 4-12 Plasmids As Vectors • pBR plasmids were developed early but are rarely used today • pUC series is similar to pBR – 40% of the DNA, including tetracycline resistance has been deleted – Cloning sites are clustered together into one area called the multiple cloning site (MCS)
- 13. 4-13 pBR322 Plasmid • pBR322 illustrates cloning methods simply – Resistance for 2 antibiotics • Tetracycline • Ampicillin – Origin of replication between the 2 resistance genes – Only 1 site for several restriction enzymes
- 14. 4-14 pBR322 Cloning Clone a foreign DNA into the PstI site of pBR322 • Cut the vector to generate the sticky ends • Cut foreign DNA with PstI also – compatible ends • Combine vector and foreign DNA with DNA ligase to seal sticky ends • Now transform the plasmid into E. coli
- 15. 4-15 Bacterial Transformation • Traditional method involves incubating bacterial cells in concentrated calcium salt solution – The solution makes the cell membrane leaky, permeable to the plasmid DNA • Newer method uses high voltage to drive the DNA into the cells in process called electroporation
- 16. 4-16 Screening Transformants • Transformation produces bacteria with: – Religated plasmid – Religated insert – Recombinants • Identify the recombinants using the antibiotic resistance – Grow cells with tetracycline so only cells with plasmid grow, not foreign DNA only – Next, grow copies of the original colonies with ampicillin which kills cells with plasmid including foreign DNA
- 17. 4-17 Screening With Replica Plating • Replica plating transfers clone copies from original tetracycline plate to a plate containing ampicillin • A sterile velvet transfer tool can be used to transfer copies of the original colonies • Desired colonies are those that do NOT grow on the new ampicillin plate
- 18. 4-18 pUC and -galactosidase Newer pUC plasmids have: – Ampicillin resistance gene – Multiple cloning site inserted into the gene lacZ’ coding for the enzyme -galactosidase • Clones with foreign DNA in the MCS disrupt the ability of the cells to make -galactosidase • Plate on media with a -galactosidase indicator (X- gal) and clones with intact -galactosidase enzyme will produce blue colonies • Colorless colonies should contain the plasmid with foreign DNA
- 19. 4-19 Directional Cloning • Cut a plasmid with 2 restriction enzymes from the MCS • Clone in a piece of foreign DNA with 1 sticky end recognizing each enzyme • The insert DNA is placed into the vector in only 1 orientation • Vector religation is also prevented as the two restriction sites are incompatible
- 20. 4-20 Summary • First generation plasmid cloning vectors include pBR322 and the pUC plasmids • pBR322 has – 2 antibiotic resistance genes – Variety of unique restriction sites for inserting foreign DNA – Most of these sites interrupt antibiotic resistance, making screening straightforward • pUC has – Ampicillin resistance gene – MCS that interrupts a -galactosidase gene • MCS facilitates directional cloning into 2 different restriction sites
- 21. 4-21 Phages As Vectors • Bacteriophages are natural vectors that transduce bacterial DNA from one cell to another • Phage vectors infect cells much more efficiently than plasmids transform cells • Clones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area
- 22. 4-22 Phage Vectors • First phage vectors were constructed by Fred Blattner and colleagues – Removed middle region – Retained genes needed for phage replication – Could replace removed phage genes with foreign DNA • Originally named Charon phage • More general term, replacement vectors
- 23. 4-23 Phage Vector Advantages • Phage vectors can receive larger amounts of foreign DNA – Charon 4 can accept up to 20kb of DNA – Traditional plasmid vectors take much less • Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle
- 24. 4-24 Cloning Using a Phage Vector
- 25. 4-25 Genomic Libraries • A genomic library contains clones of all the genes from a species genome • Restriction fragments of a genome can be packaged into phage using about 16 – 20 kb per fragment • This fragment size will include the entirety of most eukaryotic genes • Once a library is established, it can be used to search for any gene of interest
- 26. 4-26 Plaque Hybridization • Searching a genomic library requires probe showing which clone contains desired gene • Ideal probe – labeled nucleic acid with sequence matching the gene of interest
- 27. 4-27 Cosmids Cosmids are designed for cloning large DNA fragments – Behave as plasmid and phage – Contain • cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a phage head • Plasmid origin of replication permitting replication as plasmid in bacteria – Nearly all genome removed so there is room for large inserts (40-50 kb) – So little phage DNA can’t replicate, but they are infectious carrying recombinant DNA into bacterial cells
- 28. 4-28 M13 Phage Vectors • Long, thin, filamentous phage M13 • Contains: – Gene fragment with -galactosidase – Multiple cloning site like the pUC family • Advantage – This phage’s genome is single-stranded DNA – Fragments cloned into it will be recovered in single-stranded form
- 29. 4-29 M13 Cloning to Recover Single- stranded DNA Product • After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form • Use the replicative form for cloning foreign DNA into MCS • Recombinant DNA infects host cells resulting in single-stranded recombinant DNA • Phage particles, containing single-stranded phage DNA is secreted from transformed cells and can be collected from media
- 30. 4-30 Phagemids Phagemids are also vectors – Like cosmids have aspects of both phages and plasmids – Has a MCS inserted into lacZ’ gene to screen blue staining / white colonies – Has origin of replication of single-stranded phage f1 to permit recovery of single- stranded recombinant DNA – MCS has 2 phage RNA polymerase promoters, 1 on each side of MCS
- 31. 4-31 Summary • Two kinds of phage are popular cloning vectors ‑ phage ‑ Has nonessential genes removed making room for inserts - Cosmids accept DNA up to 50 kb - M13 phage - Has MCS - Produces single-stranded recombinant DNA • Plasmids called phagemids also produce single- stranded DNA in presence of helper phage • Engineered phage can accommodate inserts up to 20 kb, useful for building genomic libraries
- 32. 4-32 Eukaryotic Vectors and Very High Capacity Vectors • There are vectors designed for cloning genes into eukaryotic cells • Other vectors are based on the Ti plasmid to carry genes into plant cells • Yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) are used for cloning huge pieces of DNA
- 33. 4-33 Identifying a Specific Clone With a Specific Probe • Probes are used to identify a desired clone from among the thousands of irrelevant ones • Two types are widely used – Polynucleotides also called oligonucleotides – Antibodies
- 34. 4-34 Polynucleotide Probes Looking for a gene you want, might use homologous gene from another organism – If already cloned – Hope enough sequence similarity to permit hybridization – Need to lower stringency of hybridization conditions to tolerate some mismatches
- 35. 4-35 Control of Hybridization Stringency • Factors that promote separation of two strands in a DNA double helix: – High temperature – High organic solvent concentration – Low salt concentration • Adjust conditions until only perfectly matched DNA strands form a duplex = high stringency • Lowering these conditions lowers stringency until DNA strands with a few mismatches can hybridize
- 36. 4-36 Protein-based Polynucleotide Probes No homologous DNA from another organism? • If amino acid sequence is known, deduce a set of nucleotide sequences to code for these amino acids • Construct these nucleotide sequences chemically using the synthetic probes • Why use several? – Genetic code is degenerate with most amino acids having more than 1 nucleic acid triplet – Must construct several different nucleotide sequences for most amino acids
- 37. 4-37 Summary • Specific clones can be identified using polynucleotide probes binding to the gene itself • Knowing the amino acid sequence of the a gene product permits design of a set of oligonucleotides that encode part of the amino acid sequence • Can be a very quick and accurate means of identifying a particular clone
- 38. 4-38 cDNA Cloning • cDNA is the abbreviation for complementary DNA or copy DNA • A cDNA library is a set of clones representing as many as possible of the mRNAs in a given cell type at a given time – Such a library can contain tens of thousands of different clones
- 39. 4-39 Making a cDNA Library
- 40. 4-40 Reverse Transcriptase Primer • Central to successful cloning is the synthesis of cDNA from an mRNA template using reverse transcriptase (RT), RNA-dependent DNA polymerase – RT cannot initiate DNA synthesis without a primer – Use the poly(A) tail at 3’ end of most eukaryotic mRNA so that oligo(dT) may serve as primer
- 41. 4-41 Ribonuclease H • RT with oligo(dT) primer has made a single-stranded DNA from mRNA • Need to start to remove the mRNA • Partially degrade the mRNA using ribonuclease H (RNase H) – Enzyme degrades RNA strand of an RNA- DNA hybrid – Remaining RNA fragments serve as primers for “second strand” DNA using nick translation
- 42. 4-42 Nick Translation • The nick translation process simultaneously: – Removes DNA ahead of a nick – Synthesizes DNA behind nick – Net result moves or translates the nick in the 5’ to 3’ direction • Enzyme often used is E. coli DNA polymerase I – Has a 5’ to 3’ exonuclease activity – Allows enzyme to degrade DNA ahead of the nick
- 43. 4-43 Trailing Terminal Transferase • Don’t have the sticky ends of genomic DNA cleaved with restriction enzymes • Blunt ends will ligate, but inefficient • Generate sticky ends using terminal deoxynucleotidyl transferase (TdT), terminal transferase with one dNTP – If use dCTP with the enzyme – dCMPs are added one at a time to 3’ ends of the cDNA – Same technique adds oligo(dG) ends to vector – Generate ligation product ready for transformation
- 44. 4-44 Vector Choice • Choice based on method used to detect positive clones • Plasmid or phagemid like pUC or pBS will be used with colony hybridization and a labeled DNA probe • If phage like gt11, cloned cDNA under control of lac promoter for transcription and translation of the cloned gene and antibody screening
- 45. 4-45 Rapid Amplification of cDNA Ends • If generated cDNA is not full-length, missing pieces can be filled in using rapid amplification of cDNA ends (RACE) • Technique can be used to fill in either the missing portion at the 5’-end (usual problem) • Analogous technique can be used to fill in a missing 3’-end
- 46. 4-46 5’-RACE • Use RNA prep containing mRNA of interest and the partial cDNA • Anneal mRNA with the incomplete cDNA • Reverse transcriptase will copy rest of the mRNA • Tail the completed cDNA with terminal transferase using oligo(dC) • Second strand synthesis primed with oligo(dG)
- 47. 4-47 Summary • Make cDNA library with synthesis of cDNAs one strand at a time – Use mRNAs from a cell as templates for 1st strands, then 1st strand as template for 2nd – Reverse transcriptase generates 1st strand – DNA polymerase I generates the second strands • Give cDNAs oligonucleotide tails that base-pair with complementary tails on a cloning vector • Use these recombinant DNAs to transform bacteria • Detect clones with: – Colony hybridization using labeled probes – Antibodies if gene product translated • Incomplete cDNA can be filled in with 5’- or 3’-RACE
- 48. 4-48 4.2 The Polymerase Chain Reaction • Polymerase chain reaction (PCR) can yield a DNA fragment for cloning • PCR is: – More recently developed – Very useful for cloning cDNAs
- 49. 4-49 Standard PCR • Invented by Kary Mullis and colleagues in 1980s • Use enzyme DNA polymerase to copy a selected region of DNA – Add short pieces of DNA (primers) that hybridize to DNA sequences on either side of piece of interest – causes initiation of DNA synthesis through that area, X – Copies of both strands of X and original DNA strands are templates for next round of DNA synthesis – Selected region DNA now doubles in amount with each synthesis cycle • Special heat-stable polymerases able to work after high temperatures needed to separate strands make process “set and forget” for many cycles
- 50. 4-50 Amplifying DNA by PCR
- 51. 4-51 Using Reverse Transcriptase (RT-PCR) in cDNA Cloning • To clone a cDNA from just one mRNA whose sequence is known, use type of PCR called reverse transcriptase PCR (RT-PCR) • Difference between PCR and RT-PCR – Start with an mRNA not double-stranded DNA – Begin by converting mRNA to DNA – Next use forward primer to convert ssDNA to dsDNA – Now standard PCR continues
- 52. 4-52 RT-PCR Can Generate Sticky Ends • With care, restriction enzyme sites can even be added to the cDNA of interest • Able to generate sticky ends for ligation into vector of choice • 2 sticky ends permits directional cloning
- 53. 4-53 Real-Time PCR • Real-time PCR quantifies the amplification of the DNA as it occurs • As DNA strands separate, anneal to forward and reverse primers, and to fluorescent-tagged oligonucleotide complementary to part of one DNA strand
- 54. 4-54 Fluorescent Tags in Real-Time PCR • This fluorescent-tagged oligonucleotide serves as a reporter probe – Fluorescent tag at 5’-end – Fluorescence quenching tag at 3’-end • With PCR rounds the 5’ tag is separated from the 3’ tag • Fluorescence increases with incorporation into DNA product
- 55. 4-55 4.3 Methods of Expressing Cloned Genes Cloning a gene permits • Production of large quantities of a particular DNA sequence for detailed study • Large quantities of the gene’s product can also be obtained for further use – Study – Commerce
- 56. 4-56 Expression Vectors • Vectors discussed so far are used to first put a foreign DNA into a bacterium to replicate and screen • Expression vectors are those that can yield protein products of the cloned genes – For high level expression of a cloned gene best results often with specialized expression vectors – Bacterial vectors have a strong promoter and a ribosome binding site near ATG codon
- 57. 4-57 Fusion Proteins • Some cloning vectors, pUC and pBS, can work as expression vectors using lac promoter • If inserted DNA is in the same reading frame as interrupted gene, a fusion protein results – These have a partial - galactosidase sequence at amino end – Inserted cDNA protein sequence at carboxyl end
- 58. 4-58 Inducible Expression Vectors • Main function of expression vector is to yield the product of a gene – usually more is better • For this reason, expression vectors have very strong promoters • Prefer keep a cloned gene repressed until time to express – Large quantities of eukaryotic protein in bacteria are usually toxic – Can accumulate to levels that interfere with bacterial growth – Expressed protein may form insoluble aggregates, inclusion bodies
- 59. 4-59 Controlling the lac Promoter • lac promoter is somewhat inducible – Stays off until stimulated – Actually repression is incomplete or leaky – Some expression will still occur • To avoid this problem, express using a plasmid or phagemid carrying its own lacI repressor gene, such as pBS
- 60. 4-60 Arabinose Promoter • The hybrid trc promoter combines strength of the trp (tryptophan operon) promoter with inducibility of lac promoter • Promoter from ara operon, PBAD, allow fine control of transcription – Inducible by arabinose, a sugar – Transcription rate varies with arabinose concentration
- 61. 4-61 Tightly Controlled Promoter • Lambda () phage promoter, PL, is tightly controlled • Expression vectors with this promoter- operator system are used in host cells with temperature-sensitive repressor gene – Repressor functions are low temperatures – Raise temperature to nonpermissive temperature, the repressor doesn’t function and cloned gene is expressed
- 62. 4-62 Summary • Expression vectors are designed to yield the protein product of a cloned gene • When a lac inducer is added, cell begins to make T7 polymerase which transcribes the gene of interest • Many molecules of T7 polymerase are made, so gene is turned on to a very high level with abundant amount of protein product made
- 63. 4-63 Expression Vectors That Produce Fusion Proteins • Most vectors express fusion proteins – The actual natural product of the gene isn’t made – Extra amino acids help in purifying the protein product • Oligohistidine expression vector has a short sequence just upstream of MCS encoding 6 His – Oligohistidine has a high affinity for divalent metal ions like Ni2+ – Permits purification by nickel affinity chromatography – His tag can be removed using enzyme enterokinase without damage to the protein product
- 65. 4-65 Fusion Proteins in gt11 • This phage contains lac control region and lacZ gene • Products of gene correctly inserted will be fusion proteins with a - galactosidase leader
- 66. 4-66 Antibody Screening With gt11 • Lambda phages with cDNA inserts are plated • Protein released are blotted onto a support • Probe with antibody to protein • Antibody bound to protein from plaque is detected with labeled protein A • Partial cDNAs can be completed with RACE
- 67. 4-67 Summary • Expression vectors frequently produce fusion proteins – One part of the protein comes from coding sequences in the vector – Other part from sequences in the cloned gene • Many fusion proteins have advantage of being simple to isolate by affinity chromatography • Vector lgt11 produces fusion proteins that can be detected in plaques with a specific antiserum
- 68. 4-68 Bacterial Expression System Shortcomings • There are problems with expression of eukaryotic proteins in a bacterial system – Bacteria may recognize the proteins as foreign and destroy them – Posttranslational modifications are different in bacteria – Bacterial environment may not permit correct protein folding • Very high levels of cloned eukaryotic proteins can be expressed in useless, insoluble form
- 69. 4-69 Eukaryotic Expression Systems • Avoid bacterial expression problems by expressing the protein in eukaryotic cell • Initial cloning done in E. coli using a shuttle vector, able to replicate in both bacterial and eukaryotic cells • Yeast is suited for this purpose – Rapid growth and ease of culture – Still a eukaryote with more appropriate posttranslational modification – Secretes protein in growth medium so easy purification
- 70. 4-70 Use of Baculovirus As Expression Vector • Viruses in this class have a large circular DNA genome, 130 kb • Major viral structural protein is made in huge amounts in infected cells – Promoter for this protein, polyhedrin, is very active – These vectors can produce up to 0.5 g of protein per liter of medium – Nonrecombinant viral DNA entering cells cannot result in infectious virus as it lacks an essential gene supplied by the vector
- 72. 4-72 Animal Cell Transfection • Calcium phosphate – Mix cells with DNA in a phosphate buffer – Then solution of calcium salt added to form a precipitate – Cells take up the calcium phosphate crystals which include some DNA • Liposomes – DNA mixed with lipid to form liposomes, small vesicles with some of the DNA inside – DNA-bearing liposomes fuse with cell membrane carrying DNA inside the cell
- 73. 4-73 Summary • Foreign genes can be expressed in eukaryotic cells • These eukaryotic systems have advantages over prokaryotic ones – Made in eukaryotic cells tend to fold properly and are then soluble rather than aggregated into insoluble inclusion bodies – Posttranslational modifications are made in a eukaryotic manner
- 74. 4-74 Using the Ti Plasmid to Transfer Genes to Plants • Genes can be introduced into plants with vectors that can replicate in plant cells • Common bacterial vector promoters and replication origins are not recognized by plant cells • Plasmids are used containing T-DNA – T-DNA is derived from a plasmid known as tumor-inducing (Ti) – Ti plasmid comes from bacteria that cause plant tumors called crown galls
- 75. 4-75 Ti Plasmid Infection • Bacterium infects plant, transfers Ti plasmid to host cells • T-DNA integrates into the plant DNA causing abnormal proliferation of plant cells • T-DNA genes direct the synthesis of unusual organic acids, opines which can serve as an energy source to the infecting bacteria but are useless to the plant
- 76. 4-76 Ti Plasmid Transfers Crown Gall
- 77. 4-77 Use of the T-DNA Plasmid